Inhibition Of Carnitine Palmitoyltransferase I Research Articles

Western diet changes cardiac acyl-CoA composition in obese rats: a potential role for hepatic lipogenesis

The "lipotoxic footprint" of cardiac maladaptation in diet-induced obesity is poorly defined. We investigated how manipulation of dietary lipid and carbohydrate influenced potential lipotoxic species in the failing heart. In Wistar rats, contractile dysfunction develops at 48 weeks on a high-fat/high-carbohydrate "Western" diet, but not on low-fat/high-carbohydrate or high-fat diets. Cardiac content of the lipotoxic candidates--diacylglycerol, ceramide, lipid peroxide, and long-chain acyl-CoA species--was measured at different time points by high-performance liquid chromatography and biochemical assays, as was lipogenic capacity in the heart and liver by qRT-PCR and radiometric assays. Changes in membranes fluidity were also monitored using fluorescence polarization. We report that Western feeding induced a 40% decrease in myocardial palmitoleoyl-CoA content and a similar decrease in the unsaturated-to-saturated fatty acid ratio. These changes were associated with impaired cardiac mitochondrial membrane fluidity. At the same time, hepatic lipogenic capacity was increased in animals fed Western diet (+270% fatty acid elongase activity compared with high-fat diet), while fatty acid desaturase activity decreased over time. Our findings suggest that dysregulation of lipogenesis is a significant component of heart failure in diet-induced obesity.

Targeting malonyl CoA inhibition of mitochondrial fatty acid uptake as an approach to treat cardiac ischemia/reperfusion

Cardiovascular disease is the major cause of death and disability in the world, with ischemic heart disease accounting for the vast majority of this health problem. Current treatments for ischemic heart disease are primarily aimed at either increasing blood and oxygen supply to the heart or decreasing the heart's oxygen demand. A novel treatment strategy involves increasing the efficiency of oxygen use by the heart. During and following ischemia, the heart can become inefficient in using oxygen, due in part to an excessive use of fatty acids as a source of fuel. One potential strategy to increase cardiac efficiency is to inhibit this use of fatty acid oxidation as a fuel source, while stimulating the use of glucose oxidation as a fuel source, which allows the heart to produce energy more efficiently and reduces the acidosis associated with ischemia/reperfusion, both of which are beneficial to the heart. Malonyl CoA is a potent endogenous inhibitor of cardiac fatty acid oxidation, secondary to inhibition of carnitine palmitoyl transferase-I, the gatekeeper of mitochondrial fatty acid uptake. Malonyl CoA is synthesized in the heart by acetyl CoA carboxylase and degraded by malonyl CoA decarboxylase (MCD). Strategies aimed at increasing cardiac malonyl CoA levels, such as via inhibition of MCD, are associated with a decrease in fatty acid oxidation rates, and a parallel increase in glucose oxidation rates. This is associated with a decrease in acidosis and an improvement in cardiac function and efficiency during and following ischemia. Therefore, targeting malonyl CoA is a novel exciting approach for the treatment of cardiac ischemia/reperfusion.

Mitochondrial LCFA Oxidation, FAT/CD36 Content and CPTI Activity in Human Skeletal Muscle During Aerobic Exercise

PURPOSE: Mitochondrial fatty acid transport is a rate-limiting step in long chain fatty acid (LCFA) oxidation, and traditionally was believed to be solely regulated by carnitine palmitoyltransferase I (CPTI). Recently, it has been proposed that mitochondrial LCFA transport may involve fatty acid translocase (FAT)/CD36 in addition to CPTI, however mitochondrial FAT/CD36 has not been studied in human skeletal muscle during exercise. The present study investigated the ability of malonyl-CoA (M-CoA) to inhibit CPTI, and the presence and functional significance of FAT/ CD36 on mitochondria. We hypothesized that exercise induced increases in fatty acid oxidation would occur as a result of decreases in the sensitivity of CPTI to M-CoA and increased mitochondrial FAT/CD36 protein. METHODS: Participants cycled at ∼60% VO2peak for 120 min, and blood and ventilatory data was collected at rest and every 30 minutes during exercise. Purcutaneous needle biopsies were taken from the vastus lateralis at rest, 30 min and following 120 min. Mitochondria were immediately isolated, and used for CPTI activity measurements, palmitate oxidation rates and Western blots for FAT/CD36 protein content (n=15). RESULTS: Whole body fat oxidation rates progressively increased during exercise (p<0.05), and concomitantly M-CoA inhibition of CPTI was progressively attenuated. Compared to rest, 120 min of cycling reduced (p<0.05) the inhibition of 0.7, 2, 5 and 10 uMM-CoAby 16%, 21%, 30% and 34%, respectively. However, exercise did not alter the maximal CPTI activity. Palmitate oxidation rates in isolated mitochondria progressively increased (p<0.05) during exercise, and was positively correlated with whole body fat oxidation rates (r=0.78). Associated with the exercise induced increase in mitochondrial palmitate oxidation was a 63% increase (p<0.05) in mitochondrial FAT/CD36 protein content. Importantly, the addition of sulfo-N-succimidyl oleate, a specific inhibitor of FAT/CD36, reduced mitochondrial palmitate oxidation by ∼80%. CONCLUSIONS: The data suggests that exercise induced increases in fatty acid oxidation occur as a result of decreases in the sensitivity of CPTI to the inhibitor M-CoA and increased mitochondrial FAT/CD36 protein. It is proposed that translocation from an intracellular depot is a feasible explanation. We conclude that these two novel mechanisms are not mutually exclusive, but rather interact to influence LCFA oxidation.

Is there a single malonyl‐CoA pool in the heart for regulation of carnitine palmitoyltransferase‐I and fatty acid chain elongation

In non-lipogenic tissues malonyl-CoA serves two functions: 1) regulation of mitochondrial fatty acid oxidation via inhibition of carnitine palmitoyltransferase-I (CPT-I), and 2) providing two-carbon units for fatty acid chain elongation. In the presence of acetyl-CoA carboxylase (ACC-2) substrates acetyl-CoA, ATP-Mg2+, and bicarbonate isolated rat heart mitochondria synthesize malonyl-CoA that results in inhibition of CPT-I. Furthermore, addition of palmitoyl-CoA and NADPH/NADH to isolated intact rat heart mitochondria catalyze the incorporation of radioactive malonyl-CoA into lipid soluble products suggesting an outer mitochondrial membrane localization of the long-chain fatty acid elongation pathway. These data suggest that malonyl-CoA formed by mitochondrial ACC-2 is available for both, regulation of fatty acid oxidation and for fatty acid chain elongation. The finding with perfused heart that stable isotope labeled palmitate is chain-elongated to stearate without prior chain shortening by β-oxidation and isotope dilution as judged from the near identical isotope enrichment of palmitoyl- and stearoylcarnitine is in support of this. These data and the exclusive outer mitochondrial membrane localization of CPT-I suggests that mitochondria catalyze long-chain fatty acid elongation also in vivo. Our data raise the question about metabolic channeling of malonyl-CoA between ACC-2, CPT-I, and fatty acid chain elongation and how these processes are regulated. Supported by: DK-066107, POI AG15885, and VA Med. Res. Serv.

Mitochondrial long chain fatty acid oxidation, fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activity in human skeletal muscle during aerobic exercise

Mitochondrial fatty acid transport is a rate-limiting step in long chain fatty acid (LCFA) oxidation. In rat skeletal muscle, the transport of LCFA at the level of mitochondria is regulated by carnitine palmitoyltransferase I (CPTI) activity and the content of malonyl-CoA (M-CoA); however, this relationship is not consistently observed in humans. Recently, fatty acid translocase (FAT)/CD36 was identified on mitochondria isolated from rat and human skeletal muscle and found to be involved in LCFA oxidation. The present study investigated the effects of exercise (120 min of cycling at approximately 60% V(O2peak)) on CPTI palmitoyl-CoA and M-CoA kinetics, and on the presence and functional significance of FAT/CD36 on skeletal muscle mitochondria. Whole body fat oxidation rates progressively increased during exercise (P < 0.05), and concomitantly M-CoA inhibition of CPTI was progressively attenuated. Compared to rest, 120 min of cycling reduced (P < 0.05) the inhibition of 0.7, 2, 5 and 10 microM M-CoA by 16%, 21%, 30% and 34%, respectively. Whole body fat oxidation and palmitate oxidation rates in isolated mitochondria progressively increased (P < 0.05) during exercise, and were positively correlated (r = 0.78). Mitochondrial FAT/CD36 protein increased by 63% (P < 0.05) during exercise and was significantly (P < 0.05) correlated with mitochondrial palmitate oxidation rates at all time points (r= 0.41). However, the strongest (P < 0.05) correlation was observed following 120 min of cycling (r = 0.63). Importantly, the addition of sulfo-N-succimidyloleate, a specific inhibitor of FAT/CD36, reduced mitochondrial palmitate oxidation to approximately 20%, indicating FAT/CD36 is functionally significant with respect to LCFA oxidation. We hypothesize that exercise-induced increases in fatty acid oxidation occur as a result of an increased ability to transport LCFA into mitochondria. We further suggest that decreased CPTI M-CoA sensitivity and increased mitochondrial FAT/CD36 protein are both important for increasing whole body fatty acid oxidation during prolonged exercise.

Malonyl-CoA decarboxylase inhibition suppresses fatty acid oxidation and reduces lactate production during demand-induced ischemia

The rate of cardiac fatty acid oxidation is regulated by the activity of carnitine palmitoyltransferase-I (CPT-I), which is inhibited by malonyl-CoA. We tested the hypothesis that the activity of the enzyme responsible for malonyl-CoA degradation, malonyl-CoA decarboxlyase (MCD), regulates myocardial malonyl-CoA content and the rate of fatty acid oxidation during demand-induced ischemia in vivo. The myocardial content of malonyl-CoA was increased in anesthetized pigs using a specific inhibitor of MCD (CBM-301106), which we hypothesized would result in inhibition of CPT-I, reduction in fatty acid oxidation, a reciprocal activation of glucose oxidation, and diminished lactate production during demand-induced ischemia. Under normal-flow conditions, treatment with the MCD inhibitor significantly reduced oxidation of exogenous fatty acids by 82%, shifted the relationship between arterial fatty acids and fatty acid oxidation downward, and increased glucose oxidation by 50%. Ischemia was induced by a 20% flow reduction and beta-adrenergic stimulation, which resulted in myocardial lactate production. During ischemia MCD inhibition elevated malonyl-CoA content fourfold, reduced free fatty acid oxidation rate by 87%, and resulted in a 50% decrease in lactate production. Moreover, fatty acid oxidation during ischemia was inversely related to the tissue malonyl-CoA content (r = -0.63). There were no differences between groups in myocardial ATP content, the activity of pyruvate dehydrogenase, or myocardial contractile function during ischemia. Thus modulation of MCD activity is an effective means of regulating myocardial fatty acid oxidation under normal and ischemic conditions and reducing lactate production during demand-induced ischemia.

Myocardial protection from ischemic preconditioning is not blocked by sub-chronic inhibition of carnitine palmitoyltransferase I

Ischemic preconditioning (IP) triggers cardioprotection via a signaling pathway that converges on mitochondria. The effects of the inhibition of carnitine palmitoyltransferase I (CPT-I), a key enzyme for transport of long chain fatty acids (LCFA) into the mitochondria, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated, in isolated perfused rat hearts, whether sub-chronic CPT-I inhibition (5 days i.p. injection of 25 mg/kg/day of Etomoxir) affects I/R-induced damages and whether cardioprotection by IP can be induced after this inhibition. Effects of global ischemia (30 min) and reperfusion (120 min) were examined in hearts harvested from Control (untreated), Vehicle- or Etomoxir-treated animals. In subsets of hearts from the three treated groups, IP was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each condition was assessed by changes in infarct size as well as in myocardial contractility. Postischemic contractility, as indexed by developed pressure and d P/d t max, was similarly affected by I/R, and was similarly improved with IP in Control, Vehicle or Etomoxir treated animals. Infarct size was also similar in the three subsets without IP, and was significantly reduced by IP regardless of CPT-I inhibition. We conclude that CPT-I inhibition does not affect I/R damages. Our data also show that IP affords myocardial protection in CPT-I inhibited hearts to a degree similar to untreated animals, suggesting that a long-term treatment with the metabolic anti-ischemic agent Etomoxir does not impede the possibility to afford cardioprotection by ischemic preconditioning.

Carnitine palmitoyl transferase-I inhibition prevents ventricular remodeling and delays decompensation in pacing-induced heart failure

Experimental evidence suggests that modulation of myocardial substrate metabolism can markedly affect the progression of chronic heart failure (HF). We tested whether the inhibition of carnitine palmitoyl transferase-I (CPT-I), the enzyme regulating mitochondrial fatty acid oxidation, slows left ventricular remodeling and deterioration of function in pacing-induced HF. Normal dogs (n=9) were compared to untreated dogs with pacing-induced HF (n=9) and HF dogs treated with 65 mg/kg/day of oxfenicine (HF+Oxf, n=9), a CPT-I inhibitor. HF+Oxf reached terminal failure (LV end-diastolic pressure=25 mm Hg) 6 days later than untreated HF (P<0.05). At 28 days of pacing, hemodynamic alterations and LV dilation were significantly attenuated and the 25% decrease in LV wall thickness was completely prevented in HF+Oxf vs. untreated HF, as was the activation of matrix metalloproteinase-2 and -9, markers of tissue remodeling. Oxfenicine also prevented HF-induced transcriptional down-regulation of CPT-I, medium chain acyl-CoA dehydrogenase, GAPDH and citrate synthase, key enzymes of cardiac energy metabolism. In addition, mRNA, but not protein levels of the nuclear receptor peroxisome proliferator-activated receptor-alpha were reduced in untreated HF, while they did not change significantly in HF+Oxf, as compared to control. CPT-I inhibition early in the development of HF prevented LV wall thinning and delayed the time to end-stage failure. While these results are limited to an experimental model of disease, they nevertheless suggest that CPT-I inhibition might be effective for slowing the progression of clinical HF.

Inhibition of fatty acid synthase-dependent neoplastic lipogenesis as the mechanism of γ-linolenic acid-induced toxicity to tumor cells: an extension to Nwankwo’s hypothesis

gamma-Linolenic acid (GLA), an essential omega-6 polyunsaturated fatty acid (FA) is an attractive concept as anticancer agent because it exerts selective cytotoxic on human breast cancer cells without affecting normal cells. This selective toxicity has been identified to be due, at least in part, to the production of lipid peroxides and free radicals. Interestingly, a novel hypothesis for GLA-induced tumor cell toxicity has recently been proposed. GLA, through a molecular mechanism involving the lipogenic enzyme fatty acid synthase (FAS), coordinately interrupts the pathways that replenish the pools of metabolic intermediates in the citric acid cycle (cellular anaplerosis). First, supraphysiological concentrations of GLA inhibit glycolysis, while a cytochrome P450-dependent epoxidation of GLA generates epoxides metabolites for GLA that would mimic the inhibitory action of standard FAS inhibitors such as cerulenin and C75. Second, GLA-epoxide inhibits FAS activity, thus resulting in the accumulation of cytosolic malonyl-CoA which, in turn, inhibits carnitine palmitoyl transferase I (CPT-I) and prevents FA oxidation. The recent characterization of GLA as a novel regulator of FAS expression in breast cancer cells supports and further expands this hypothesis, and directly involves FAS-dependent de novo fatty acid synthesis as the mechanism of GLA-induced toxicity to tumor cells. We hypothesize that, at low (physiological) concentrations, the inhibitory effect of GLA on FAS-regulated breast cancer cell survival is not specific and is due to cell toxicity caused by lipid peroxidation. Taking into account that the inhibitory effect of FAs on the expression of FAS in cultured hepatocytes has been shown to be related to a non-specific peroxidative mechanism, a similar GLA-dependent FAS regulatory mechanism involving peroxidative products may occur in normal and neoplastic tissues. At high (supraphysiological) concentrations of GLA, the specific downregulation of FAS gene expression leads to accumulation of the substrate for FAS, malonyl-CoA, that, as a result of FAS blockade, continue to be generated by the rate-limiting enzyme of the fatty acid biosynthetic pathway acetyl-CoA carboxilase, which is not inhibited in the absence of FAS-catalyzed long chain endogenous fatty acids. Physiologically, the elevated levels of malonyl-CoA occurring during FA biosynthesis reduce FA oxidation to prevent a futile cycle of simultaneous FA synthesis and degradation. Paradoxically, high-dose GLA treatments of FAS-overexpressing breast cancer cells will promote malonyl-CoA-induced inhibition of CPT-I and FA oxidation, thus precipitating an energy crisis that triggers decreased proliferation or apoptotic cell death. In summary, this working model presents the concept that the breast cancer adaptation in FAS expression can be exploited to develop GLA-based dietary interventions aimed at altering the FA synthesis pathway, which appears to be linked to neoplastic transformation and is associated with tumor virulence and adverse clinical outcome in a subset of human breast carcinomas.

Expression of three carnitine palmitoyltransferase-I isoforms in 10 regions of the rat brain during feeding, fasting, and diabetes

Inhibition of carnitine palmitoyltransferase-I (CPT-I) activity in the brain has been shown to decrease food intake in rats. We examined the expression of mRNA encoding all three known CPT-I isoforms (α, β, and γ) in 10 different major regions of the rat brain in normal, chow-fed rats, in fasting rats, and in insulin-dependent diabetic rats. Compared with the effects of fasting and diabetes on CPT-I mRNA in the liver and heart, there was either less effect or no effect depending on the particular brain region examined. These results suggest that the regulation of CPT-I mRNA levels is different in the brain than in other tissues. A surprising result of this study was the discovery of very high, unique expression of CPT-Iβ (the muscle isoform) in the cerebellum.

Beta-Hydroxybutyrate inhibits myocardial fatty acid oxidation in vivo independent of changes in malonyl-CoA content.

This study tested the hypothesis that an acute infusion of beta-hydroxybutyrate inhibits myocardial fatty acid uptake and oxidation in vivo. Anesthetized pigs were untreated (n = 6) or treated with an intravenous infusion of fat emulsion (n = 7) to elevate plasma free fatty acid levels. A third group received fat emulsion plus an intravenous infusion of beta-hydroxybutyrate (25 micromol.kg-1.min-1; n = 7) for 60 min. All animals received a continuous infusion of [3H]palmitate, and myocardial fatty acid oxidation was measured from the cardiac production of 3H2O. Plasma free fatty acid concentrations were elevated in the fat emulsion group (0.77 +/- 0.11 mM) compared with the untreated group (0.15 +/- 0.03 mM), which resulted in greater myocardial free fatty acid oxidation. In contrast, the group receiving beta-hydroxybutyrate in addition to fat emulsion had elevated beta-hydroxybutyrate concentration (0.87 +/- 0.11 vs. 0.04 +/- 0.01 mM), but suppressed fatty acid oxidation (0.053 +/- 0.013 micromol.g-1.min-1) (P < 0.05) compared with the fat emulsion group (0.116 +/- 0.029 micromol.g-1.min-1). There were no differences among the three groups in the tissue content for malonyl-CoA, acetyl-CoA, or free CoA or the activity of acetyl-CoA carboxylase; thus the inhibition of fatty acid oxidation by elevated beta-hydroxybutyrate did not appear to be due to malonyl-CoA inhibition of carnitine palmitoyl transferase-I or to an increase in the acetyl-CoA-to-free CoA ratio. In conclusion, fatty acid uptake and oxidation is blocked by an infusion of beta-hydroxybutyrate; this effect was not due to elevated myocardial malonyl-CoA content.

Differential effects of peroxisome proliferator-activated receptor activators on the mRNA levels of genes involved in lipid metabolism in primary human monocyte-derived macrophages

Peroxisome proliferator-activated receptors (PPARs) are key regulators of macrophage lipid metabolism. We compared the effects of three PPAR activators (bezafibrate, fenofibrate, and troglitazone) on the mRNA levels of genes involved in lipid metabolism in primary human macrophages and macrophage-derived foam cells. Treatment of human macrophages for 24 hours with 100 μmol/L bezafibrate, a nonselective drug that activates the 3 PPAR subtypes (PPARα, PPARβ/δ, and PPARγ), caused an 87% (P <.01) and a 230% rise in CD36 and adipocyte fatty acid-binding protein (aP2) mRNA levels, respectively, whereas the expressions of PPARγ, PPARα, acyl-CoA oxidase, carnitine palmitoyltransferase I (CPT-I), adenosine triphosphate (ATP)-binding cassette transporter 1 (ABCA1), neutral cholesteryl ester hydrolase, and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) were not modified. However, treatment with selective PPARα (fenofibrate at 100 μmol/L) and PPARγ (troglitazone at 5 μmol/L) activators had different effects. Fenofibrate increased PPARα (62%, P <.05) and LOX-1 (180%, P <.05) mRNA levels; and troglitazone upregulated CPT-I expression (75%, P <.05). When the effects of these drugs were assessed in macrophage-derived foam cells, we found that troglitazone caused a 134% (P <.05) and a 66% (P <.01) rise in ABCA1 and CPT-I mRNA levels, respectively, whereas the 3 drugs significantly increased aP2 transcripts (about 100% induction). Given that troglitazone treatment resulted in the upregulation of genes involved in the mitochondrial β-oxidation of fatty acids (CPT-I) and in the reverse-cholesterol-transport pathway (ABCA1), we subsequently determined whether these changes affected intracellular cholesterol ester accumulation. In macrophage-derived foam cells a significant reduction (32%, P <.01) was observed in intracellular cholesterol accumulation after troglitazone, but not after bezafibrate or fenofibrate treatment. Since CPT-I inhibition promotes cholesterol incorporation into cholesteryl esters in macrophages, study is now needed on whether CPT-I induction by troglitazone may reduce the availability of fatty acids for synthesizing cholesterol esters, leading to less foam cell formation. © 2003 Elsevier Inc. All rights reserved.

Fatty acids are important for the Frank-Starling mechanism and Gregg effect but not for catecholamine response in isolated rat hearts.

In some pathophysiological conditions myocardial metabolism can switch from mainly long chain fatty acid (LCFA) oxidation to mainly glucose oxidation. Whether the predominant fatty acid or glucose oxidation affects cardiac performance has not been defined. In a buffer perfused isovolumetrically contracting rat heart, oxidation of endogenous pool LCFA was avoided by inhibiting carnitine-palmitoyl-transferase I (CPT-I) with oxfenicine (2 mM). In order to restore fatty acid oxidation, hexanoate (1 mM), which bypasses CPT-I inhibition, was added to the perfusate. Three groups of hearts were subjected to either an increase in left ventricular volume (VV, +25%) or an increase in coronary flow (CF, +50%), or inotropic stimulation with isoproterenol (10(-8) and 10(-6) m). The increase in VV (the Frank-Starling mechanism) increased rate-pressure product (RPP) by 21 +/- 2% under control conditions, but only by 6 +/- 2% during oxfenicine-induced CPT-I inhibition. The contractile response to changes in VV recovered after the addition of hexanoate. Similar results were obtained in hearts, in which an increase in CF was elicited (the Gregg phenomenon). Isoproterenol caused a similar increase in contractility regardless of the presence of oxfenicine or hexanoate. In all groups, a commensurate increase in oxygen consumption accompanied the increase in contractility. The fatty acid oxidation is necessary for an adequate contractile response of the isolated heart to increased pre-load or flow, whereas the inotropic response to adrenergic beta-receptor stimulation is insensitive to changes in substrate availability.

Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms.

The prevailing concept of the heart’s response to changes in its environment is a complex network of inter-connecting signal transduction cascades.1 In such a scheme, the focus is on communication of various cell surface receptors, heterotrimeric G-proteins, protein kinases, and transcription factors.2–4⇓⇓ Diabetes is a disorder of metabolic dysregulation. At first glance it appears that metabolism and the metabolic consequences of diabetes do not fit into this signal-response coupling scheme. Two questions arise. First, is metabolism simply an “effect” rather than a “cause” of adaptation? Second, is metabolism only a by-product of signal transduction-induced adaptation, allowing equilibrium (and therefore maintenance of function) in the presence of the other adaptational responses? An alternative is to take a new, less restricted view of metabolism. Beyond its stereotypical function as a provider of ATP, alterations in metabolic flux within the cell create essential signals for the adaptation of the heart to situations such as diabetes. This concept is novel for the heart, but has already been considered in the liver. Like the phosphorylation events occurring in signal transduction cascades, changes in metabolic flux are extremely rapid. For example, translocation of GLUT4 to the cell surface in response to insulin occurs within a second.5 We have previously found that increases or decreases in workload also change metabolic fluxes in seconds.6,7⇓ Therefore, changes in metabolites are rapid enough to allow them to act as signaling molecules. Many of these acute changes in metabolic flux are brought about by the same signal transduction cascades believed to be involved in the adaptation of the heart to changes in its environment. Phosphatidylinositol 3-kinase, Ca2+, and protein kinase C, all of which play a role in cardiac adaptation, regulate metabolism in the heart.8,9⇓ Metabolic signals therefore provide a …

Uncoupling protein-3 mRNA up-regulation in C2C12 myotubes after etomoxir treatment

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of C2C12 myotubes for 24 h with 40 μM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated uncoupling protein 3 (UCP-3) mRNA levels (2-fold induction), whereas UCP-2 mRNA levels were not modified. Etomoxir treatment also caused a 2.5-fold induction in M-CPT-I (muscle-type CPT-I) mRNA levels. In contrast, other well-known peroxisome proliferator-activated receptor α (PPARα) target genes, such as acyl-CoA oxidase and medium-chain acyl-CoA dehydrogenase, were not affected, suggesting that this transcription factor was not involved in the effects of etomoxir. Since it has been reported that CPT-I inhibition by etomoxir leads to a further increase in ceramide synthesis, we test the possibility that ceramides were involved in the changes reported. Similarly to etomoxir, addition of 20 μM C 2-ceramide to C2C12 myotubes for 3, 6 and 9 h resulted in increased UCP-3 and M-CPT-I mRNA levels. These results indicate that the effects on UCP-3 mRNA levels could be mediated by increased ceramide synthesis.

Etomoxir, Sodium 2-[6-(4-Chlorophenoxy)hexyl]oxirane-2-carboxylate, Up-Regulates Uncoupling Protein-3 mRNA Levels in Primary Culture of Rat Preadipocytes

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of primary culture of rat preadipocytes for 24 h with 40 μM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated UCP-3 mRNA levels (3.6-fold induction), whereas changes in UCP-2 mRNA levels were not significant. As a consequence of increased UCP-3 expression, a fall in the mitochondrial membrane potential was detected by flow cytometry. Etomoxir treatment modified neither L-CPT-I (liver-type) nor PPARα mRNA levels in preadipocytes. In contrast, mRNA expression of acyl-CoA oxidase (ACO), the rate-limiting enzyme of peroxisomal fatty acid β-oxidation, whose transcription is controlled by PPARα, was significantly induced (1.3-fold induction, P = 0.015). These findings suggest that the effects of etomoxir were mediated by PPARα. Since it has been reported that the intracellular accumulation of lipids following the inhibition of CPT-I by etomoxir leads to a PPARα-mediated metabolic response that increases the expression of genes involved in alternate fatty acid oxidation pathways, these results seem to implicate UCP-3 in this protective metabolic response. It remains to be studied whether reductions in the expression of UCP-3 could compromise this response, giving rise to lipotoxic effects on cells.

Loss of response of carnitine palmitoyltransferase I to okadaic acid in transformed hepatic cells

The specific activity of carnitine palmitoyltransferase I (CPT-I) was similar in mitochondria isolated from rat Fao and human HepG2 hepatoma cells and from rat hepatocytes, but almost twofold higher in permeabilized hepatoma cells than in permeabilized hepatocytes. Short-term exposure to okadaic acid induced a ca. 80% stimulation of CPT-I in hepatocytes, whereas no significant response of the enzyme from hepatoma cells was evident. Thus, the high CPT-I activity displayed by hepatoma cells may be reached by hepatocytes upon challenge to okadaic acid. Reconstitution experiments with purified mitochondrial and cytoskeletal fractions showed that the cytoskeleton of hepatocytes produced a more remarkable inhibition of CPT-I than the cytoskeleton of Fao cells. The present data may be explained by a disruption of interactions between CPT-I and cytoskeletal components in tumor cells that may be involved in the okadaic acid-induced activation of hepatic CPT-I as previously suggested.

Evidence that the AMP-activated protein kinase stimulates rat liver carnitine palmitoyltransferase I by phosphorylating cytoskeletal components

The activity of hepatic carnitine palmitoyltransferase I (CPT-I) may be modulated by interactions with cytoskeletal components [Velasco et al. (1998) J. Biol. Chem. 273, 21497–21504]. We have studied whether the AMP-activated protein kinase (AMPK) is involved in this process. AMPK stimulated CPT-I in permeabilized hepatocytes but not in isolated liver mitochondria. In addition, AMPK abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytoskeletal fraction. These two effects of AMPK were not evident when the kinase was inactivated by pretreatment with protein phosphatase 2C. Cytokeratins 8 and 18 were phosphorylated by AMPK in vitro and by incubation of intact hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside, a cell-permeable activator of AMPK. These results provide the first evidence that AMPK stimulates CPT-I by direct phosphorylation of cytoskeletal components.

Malonyl-CoA-independent Acute Control of Hepatic Carnitine Palmitoyltransferase I Activity: ROLE OF Ca2+/CALMODULIN-DEPENDENT PROTEIN KINASE II AND CYTOSKELETAL COMPONENTS

The mechanism of malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I (CPT-I) activity was investigated. In a first series of experiments, the possible involvement of the cytoskeleton in the control of CPT-I activity was studied. The results of these investigations can be summarized as follows. (i) Very mild treatment of permeabilized hepatocytes with trypsin produced around 50% stimulation of CPT-I activity. This effect was absent in cells that had been pretreated with okadaic acid (OA) and seemed to be due to the action of trypsin on cell component(s) distinct from CPT-I. (ii) Incubation of intact hepatocytes with 3, 3'-iminodipropionitrile, a disruptor of intermediate filaments, increased CPT-I activity in a non-additive manner with respect to OA. Taxol, a stabilizer of the cytoskeleton, prevented the OA- and 3, 3'-iminodipropionitrile-induced stimulation of CPT-I. (iii) CPT-I activity in isolated mitochondria was depressed in a dose-dependent fashion by the addition of a total cytoskeleton fraction and a cytokeratin-enriched cytoskeletal fraction, the latter being 3 times more potent than the former. In a second series of experiments, the possible link between Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII) and the cytoskeleton was studied in the context of CPT-I regulation. The data of these experiments indicate that (i) purified Ca2+/CM-PKII activated CPT-I in permeabilized hepatocytes but not in isolated mitochondria, (ii) purified Ca2+/CM-PKII abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytokeratin-enriched fraction, and (iii) the Ca2+/CM-PKII inhibitor KN-62 prevented the OA-induced phosphorylation of cytokeratins in intact hepatocytes. Results thus support a novel mechanism of short-term control of hepatic CPT-I activity which may rely on the cascade Ca2+/CM-PKII activation --> cytokeratin phosphorylation --> CPT-I de-inhibition.

Diabetes and proteolysis: effects on carnitine palmitoyltransferase-I and malonyl-CoA binding.

Malonyl-CoA binding and malonyl-CoA inhibition of carnitine palmitoyltransferase-I (CPT-I) were measured in hepatic mitochondria from normal and diabetic rats and in protease-treated mitochondria from fed rats to test the hypothesis that proteolysis represents a mechanism by which diabetes produces changes in the sensitivity of CPT-I to inhibition by malonyl-CoA. As in diabetes, protease treatment increased the apparent Ki values for malonyl-CoA. Palmitoyl-CoA greatly diminished malonyl-CoA specific binding in the mitochondrial system being studied, suggesting strong competition at the malonyl-CoA binding site. Proteolysis decreased capacity for specific binding of malonyl-CoA by 60-80%, but it had no effect on binding affinity. In contrast, the decreased specific binding of malonyl-CoA seen in the diabetic state is accompanied by increased binding affinity. Furthermore, observed Kd values differed from Ki values by a factor of 10 or more, suggesting that measured Kd and Ki may represent different ligand-protein complexes. These data suggest that alterations in inhibition of CPT-I by malonyl-CoA occurring in the diabetic state may involve mechanisms other than simple proteolytic removal of malonyl-CoA binding sites.